May 04, 2026
Comet Protocol for FFPE slides - Benchmarking Project SenNet
- Nicola Neretti1,
- Samantha Madnick1,
- Maria H Pedersen2
- 1Brown University;
- 2New York Genome Center
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Nicola Neretti, Samantha Madnick, Maria H Pedersen 2026. Comet Protocol for FFPE slides - Benchmarking Project SenNet. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6j52dvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 27, 2026
Last Modified: May 04, 2026
Protocol Integer ID: 315824
Keywords: iterative indirect immunofluorescence imaging (4i), FFPE, tissue section, antibody elution, protein, using iterative indirect immunofluorescence imaging, iterative indirect immunofluorescence imaging, indirect immunofluorescence imaging, eluting antibodies between imaging round, multiplexed protein measurement, protein targets within the same tissue section, protocol for ffpe slide, imaging round, ffpe slide, imaging, protein target, eluting antibody, secondary antibody, antibody, ffpe, benchmarking project sennet, comet protocol for ffpe slide, comet protocol, comet machine, comet machine from lunaphore, benchmarking project sennet this protocol
Funders Acknowledgements:
NIH U54 SenNet
Grant ID: 1U54AG076040
Abstract
This protocol describes a workflow for highly multiplexed protein measurements using the Comet machine from Lunaphore. This method is optimized for formalin-fixed paraffin-embedded (FFPE) tissue sections and enables repeated cycles of staining, imaging and antibody elution using conventional primary and secondary antibodies. By sequentially eluting antibodies between imaging rounds, this approach allows the detection of a large number of protein targets within the same tissue section while preserving spatial context. This protocol was adapted from methods developed in the laboratory of Dr Joao Passos made for iterative indirect immunofluorescence imaging (4i).
Materials
- Ice
- SuperFrost Plus slides
- Histoclear
- Ethanol (100%, 90%, 70%)
- Distilled water
- Coplin jars
- Deionized water
- Citrate buffer 1x working solution
- 10x Citrate buffer pH 6.0 (Abcam AB64214)
- Hot plate or microwave
- Heat resistant gloves
- Aluminum foil
- PBS
Sectioning of paraffin blocks
Samples are placed on ice for at least 20 minutes
Trim block until sample is exposed
Sections are cut at 10um
Sections are laid on a water bath that is between 40-45˚C to unfold wrinkles
Sections are picked up onto a SuperFrost Plus slide
Slides are air dried at room temperature for 30 minutes
Slides are placed in an oven at 60˚C overnight
Deparaffinization and Hydration
Deparaffinize and hydrate tissue sections through Histoclear 2×5 min (in fume hood), 100% ethanol 2×5 min, 90% ethanol 5 min, 70% ethanol 5 min, distilled water 2×5 min. These steps are performed in Coplin Jars
Steamer assembly
Fill the water basin with deionized water
Place the plastic steaming compartment atop the water basin
Place an empty Coplin jar in the steaming compartment
Turn on the steamer until steam is visible escaping the vent. Keep the lid closed
Using a hot plate or microwave, boil ~75 ml of citrate buffer 1x working solution
Citrate buffer 1x working solution
7ml 10x Citrate buffer pH 6.0 (Abcam AB64214)
Add 63 ml distilled water
Using heat resistant gloves, carefully pour the boiling sodium citrate buffer into Coplin jar within the steaming compartment
Add slides to Coplin jar, cover in aluminum foil and close lid
Incubate for 10 minutes
Turn off steamer and remove lid. Make sure citrate buffer solution is still covering entire tissue
Let slides to cool down in the citrate buffer 20-30 minutes at room temperature (or to cool it faster place on ice until buffer is cold)
Wash sections in distilled water 2×5 min
Wash sections for 5 min PBS on shaker
Comet run
Prepare the reagents according to the reagents protocol attached and run the Comet according to the manufactures guideline. 
Comet_reagent_preparation_skin.pdf Comet_reagent_preparation_lung.pdf
Comet_reagent_preparation_skin.pdf 
Comet_reagent_preparation_lung.pdf